1. Field of the Invention
The present invention relates generally to the fields of medical imaging, analysis, monitoring and diagnostics. More particularly, it provides apparatuses and methods for analyzing proteins in samples. Even more particularly, it may be used in the direct profiling of diseased tissue by mass spectrometry; this, in turn, may be used for the assessment of disease classification, development and treatment.
2. Description of Related Art
Every year about 1.5 million Americans are diagnosed with cancer. Tragically about 500,000 people die of the disease every year. Cancer can affect people in a variety of different ways: about 11% of cancer patients are diagnosed with colorectal cancer, 15% with prostate cancer, 20% with lung cancer, 15% with breast cancer, and 2% with brain cancer. The ability to effectively identify specific tumor markers in proliferating areas of the tumors would therefore be a beneficial step in diagnosing, monitoring, analyzing, and treating such tumors and, in general, a wide variety of other ailments.
Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is an analytical technique having high sensitivity, ease of use, and compatibility as an effective off-line method for different types of sample analysis. Static sampling systems using MALDI MS have demonstrated extremely high sensitivities, as illustrated by attomole sensitivity for the analysis of peptides contained in complex physiological salt solutions. Further, matrix precoated cellulose targets have been used to analyze 100% aqueous samples without the need of further treatment with organic solvents.
Several reports have described the use of MALDI for the analysis of specific peptides in whole cells. Several papers describe the analysis of some neuropeptides directly in single neurons of the mollusk Lymnaea stagnalis. Isolated neurons were ruptured, mixed with small volumes of matrix, and analyzed. The ability of MALDI MS to be used to elucidate some of the metabolic processing involved in neuropeptide production from precursor peptides has also been demonstrated. Also, a single neuron from Aplypsia californica was analyzed for several specific neuropeptides using a procedure involving removal of excess salt by rinsing with matrix solution.
A considerable amount of work has been described for use of secondary ion mass spectrometry (SIMS) for the spatial arrangement of elements in surfaces of samples including biological tissue and organic polymers. In addition, there have been recent efforts to apply the SIMS technique to organic compounds and metabolites in biological samples. At least one report describes conditions for generating secondary ion mass spectra from samples with choline chloride and acetylcholine chloride deposited onto specimens of porcine brain tissue. Samples were then exposed to a primary ion beam of massive glycerol clusters. Images generated from the spacially arranged SIMS spectra were obtained that reflected the identity and location of the spiked analytes.
U.S. Pat. No. 5,808,300 which is incorporated herein by reference, describes a method and apparatus for imaging biological samples with MALDI MS. Its techniques can be used to generate images of samples in one or more m/z pictures, providing the capability for mapping the concentrations of specific molecules in X, Y coordinates of the original biological sample. Analysis of a biological sample can be carried out directly or to an imprint of the sample. The image attained in the analysis can be displayed in individual m/z values as a selected ion image, as summed ion images, or as a total ion image. The imaging process may also be applied to other separation techniques where a physical track or other X, Y deposition process is utilized.
U.S. Pat. No. 5,272,338 which is incorporated herein by reference, describes a specific instrument setup using a liquid metal ion source to ionize a sample in a mass spectrometer, and then a laser beam to irradiate the ejected molecules and resonantly ionize them. The method involves a technique commonly referred to as SIMS/cross beam laser ionization.
U.S. Pat. Nos. 5,372,719 and 5,453,199 which are incorporated herein by reference, disclose techniques for preparing a chemically active surface so that when a sample is exposed to this surface, a chemical image of the sample is deposited on the surface. The disclosed methods involve the separation of molecules by sorbents.
U.S. Pat. No. 5,607,859 which is incorporated herein by reference, describes a method for the MS determination of highly polyionic analytes by the interaction of oppositely charged molecules.
U.S. Pat. No. 5,569,915 which is incorporated herein by reference, discloses an MS instrument for fragmenting molecules in the gas phase.
U.S. Pat. No. 5,241,569 which is incorporated herein by reference, describes neutron activation analysis for detecting gamma rays and beta-electrons from radioactively labeled samples. This technique may be used to locate elements in a sample.
Although techniques referenced above show at least a degree of utility in performing certain types of general analysis, shortcomings remain. For instance, current techniques used for rapid decisions in the clinical laboratory often involve the analysis of frozen sections using light microscopy. Such techniques, however, are, at times, non-specific and inaccurate. More importantly, conventional technology does not involve techniques that effectively employ the capability of MALDI MS to analyze and effectively depict a quantity of molecules of interest with a specific molecular mass or within a selected molecular mass window or as a function of their position on the test sample. Further, conventional technology does not employ MALDI MS techniques to effectively analyze proteins in clinical samples. Still further, conventional technology does not use MALDI MS techniques to perform direct profiling of diseased tissue, which in turn may be used for the assessment of disease classification, development and treatment.